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SPECIATION AND BIOGEOCHEMICAL PROCESSES OF TRACE ELEMENTS IN A NORTHEASTERN ATLANTIC REGION:

I. Levels and fluxes of trace elements in the Vigo Ria.

II. Dissolved copper speciation in the freshwater discharges and estuarine mixing of a ria system.

III. Impact of the Prestige oil spill on dissolved trace metal levels in Galician coastal and offshore waters.

Juan SANTOS ECHEANDÍA

Vigo, April 2009

2009

Juan

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PhD THESIS

Memoria presentada por Juan Santos Echeandía para optar al título de “Doctor Europeus” por la Universidade de Vigo

SPECIATION AND BIOGEOCHEMICAL PROCESSES OF TRACE ELEMENTS IN A NORTHEASTERN ATLANTIC

REGION: I. Levels and fluxes of trace elements in the Vigo Ria

II. Dissolved copper speciation in the freshwater discharges

and estuarine mixing of a ria system. III. Impact of the Prestige oil spill on dissolved trace metal

levels in Galician coastal and offshore waters.

Juan Santos Echeandía

PhD THESIS

Vigo, April 2009

Departamento de Química Analítica y Alimentaria

Facultad de Ciencias (UVigo)

Grupo de Biogeoquímica Marina Instituto de Investigaciones Marinas de Vigo

(IIM, CSIC)

SPECIATION AND BIOGEOCHEMICAL PROCESSES OF TRACE ELEMENTS IN A NORTHEASTERN ATLANTIC

REGION: I. Levels and fluxes of trace elements in the Vigo Ria

II. Dissolved copper speciation in the freshwater discharges

and estuarine mixing of a ria system. III. Impact of the Prestige oil spill on dissolved trace metal

levels in Galician coastal and offshore waters.

Tesis doctoral presentada en el Departamento de Química Analítica y Alimentaria de la Universidade de Vigo Memoria presentada por Juan Santos Echeandía para optar al título de “Doctor Europeus” por la Universidade de Vigo

Realizada bajo la dirección de

Dr. Ricardo Prego Reboredo Dr. Antonio Cobelo García

Y la tutoría de

Dr. Óscar Nieto Palmeiro

Vigo, 30 de Abril de 2009

Departamento de Química Analítica y Alimentaria

Facultad de Ciencias (UVigo)

Grupo de Biogeoquímica Marina Instituto de Investigaciones Marinas de Vigo

(IIM, CSIC)

Ricardo Prego Reboredo, Profesor de Investigación y Antonio Cobelo García,

Científico Titular, ambos del Consejo Superior de Investigaciones Científicas

(CSIC) en el Instituto de Investigaciones Marinas de Vigo, y Óscar Nieto Palmeiro, Profesor Titular del Departamento de Química Analítica y Alimentaria de

la Universidad de Vigo:

CERTIFICAN QUE:

La presente memoria titulada SPECIATION AND BIOGEOCHEMICAL

PROCESSES OF TRACE ELEMENTS IN A NORTHEASTERN

ATLANTIC REGION. I. Levels and fluxes of trace elements in the Vigo Ria; II. Dissolved copper speciation in the freshwater

discharges and estuarine mixing of a Ria system; III. Impact of the

Prestige oil spill on dissolved trace metal levels in Galician coastal

and offshore waters, para optar al Grado de “Doctor Europeus” que

presenta Juan Santos Echeandía ha sido realizada bajo nuestra

dirección en el Grupo de Biogeoquímica del IIM (CSIC) y tutoría en la

Facultad de Ciencias (Universidad de Vigo).

Considerando que representa trabajo de Tesis, autorizan su

presentación ante la Comisión de Doctorado de la Universidad de Vigo.

Y para que así conste y surta los efectos oportunos, firmamos el

presente certificado en Vigo a 30 de Abril de 2009.

Los directores

D. Ricardo Prego Reboredo D. Antonio Cobelo García

Tutor El doctorando

D. Óscar Nieto Palmeiro Juan Santos Echeandía

A mis padres y hermana A Lucía

“O verdadeiro heroísmo consiste en trocar os anceios en realidades, as ideias en feitos” (Castelao, 1886-1950)

AGRADECIMIENTOS

Cinco años después de haber iniciado este largo camino puedo ponerme a escribir los agradecimientos con la tranquilidad y el orgullo de haber superado uno tras otro tantos obstáculos como surgieron hasta la finalización de mi Tesis Doctoral. No ha sido un camino fácil pero un mar tranquilo no hace habilidoso al marinero. Suelen decir que “lo que no te mata te hace más fuerte” y puedo asegurar que éste periodo de mi vida me ha formado tanto científica como personalmente aunque, como bien dice mi padre siempre se puede mejorar en todo y uno no debe apoltronarse nunca porque la vida es un continuo aprendizaje y hay que disfrutar de ello.

Ni que decir tiene que uno no podría haber llegado aquí sin la ayuda desinteresada o no de gran cantidad de gente entre los que incluyo familiares, amigos, compañeros, profesores, los cuales han servido de apoyo en los momentos de desmotivación o bajón surgidos durante la realización de ésta memoria. A todos ellos va dirigido este agradecimiento general con el que pretendo que nadie se sienta ofendido ni olvidado por no haber sido mencionado.

A continuación, deseo agradecer más personalmente a una serie de personas que han contribuido en una parte importante en la realización de esta Tesis Doctoral,

Al Dr. Ricardo Prego, quien me tendió la mano y me acogió bajo su dirección tras mi llegada al Grupo de Biogeoquímica Marina cuando aún no me había ni siquiera Licenciado en Ciencias del Mar. Me siento afortunado por haberte tenido como director y por la gran calidad y cantidad de ciencia que me has transmitido en estos 5 años. Gracias por haber confiado en mí y haberme animado siempre, espero no haberte defraudado.

Al Dr. Antonio Cobelo-García, cuya irrupción como co-director de mi Tesis supuso un soplo de aire fresco y complementario en mi formación. Agradezco todas y cada una de las charlas científicas que hemos compartido en diversos ambientes y países, desde el laboratorio o el despacho, hasta el pub pasando por

los coffee-breaks a media tarde en la azotea del IIM. En ciencia no se aprende y se avanza únicamente en el despacho y/o laboratorio.

Al Dr. Óscar Nieto Palmeiro, mi tutor en la Universidad de Vigo y gran valedor a mi llegada al Grupo de Biogeoquímica del IIM. Muchas gracias por la ayuda logística, burocrática, científica y moral prestada por tu parte desde el CUVI.

A mi padre, sin ninguna duda mi ejemplo y espejo en el que mirarme desde que tengo uso de razón en esta vida. A pesar de que el inconformismo es una de las cosas más valiosas que he aprendido de tí, me conformo con llegar a ser en mi campo profesional la mitad de lo que tú has llegado a ser en el tuyo.

A mi madre, por haberme inculcado la meticulosidad, el tesón, el orden y la previsión (entre otras muchas virtudes) en cada una de las cosas que se hacen en la vida. Cuantos quebraderos de cabeza y disgustos evitados gracias a ello.

Gracias a los dos por haber confiado siempre en mí y por habérmelo dado todo, estuviera o no a vuestro alcance. Siempre he intentado responder a vuestras expectativas a pesar de que ha habido épocas peores superadas gracias a vuestro apoyo y valiosos consejos. Parece que tanto “sostener” muestras ha dado sus frutos. Espero que estéis tan orgullosos de mí como lo estoy yo de vosotros.

Hermana, qué emoción poderte agradecer dentro de la memoria de mi Tesis todos los momentos compartidos, tanto buenos como malos, pero que a fin de cuentas nos han hecho ser como uña y carne en la distancia (esperemos que el AVE no tarde mucho en llegar). Que sepas que estoy muy orgulloso de tí. Josean, no te creas que te vas a librar de aparecer en los agradecimientos. Sé lo mucho que me aprecias, tanto como yo a tí. Ya vas a poder decir a tus amigos que el hermano de tu novia, “el científico”, va a convertirse en Doctor en Ciencias del Mar.

A Lucía, por las horas robadas, por toda la paciencia y comprensión que ha tenido conmigo en todo este tiempo de constantes idas y venidas (sé que en estos años me he parecido bastante al Guadiana), y por haber aguantado cada tarde mis sermones contando algún problema, queja o preocupación tras mi llegada a casa después de un largo día en el laboratorio. Gracias por estar siempre a mi lado me encuentre donde me encuentre.

A todos los compañeros del laboratorio dentro del Grupo de Biogeoquímica Marina por todos los buenos momentos compartidos y en especial a Clemente, Patricia, Unai, Judith, Iñigo, Paula, Natalia, Ana Garci, Azucena y Ana Virginia (seguro que me olvido de alguien) así como toda la gente de prácticas que ha pasado a lo largo de estos años por el Grupo.

Al Dr. Stan van den Berg de la Universidad de Liverpool por haberme recibido en su laboratorio y ayudado a dar un paso más dentro de las aplicaciones de la voltamperometría en el campo de los metales traza. No me olvido tampoco de la gente (Luis, Conrad, Gianluca y Pascal) con la que compartí buenos ratos en el laboratorio de Electroquímica y de los cuales me llevo muy buenos recuerdos.

Al Dr. Geoff Millward de la Universidad de Plymouth por haberme tutelado durante mi estancia en su laboratorio y haberme aportado una inestimable ayuda en la compresión de numerosos procesos sobre los metales en estuarios. Nunca pensé que alguien pudiera tener la misma ilusión y ambición por su trabajo que el primer día después de haber estado en el top de la ciencia durante tantos años. Sin duda todo un ejemplo. También he de mencionar aquí a todos los compañeros de oficina y laboratorio, técnicos y profesores (Maeve, los Andies, Malcolm, Jose,

Juan, Estela, Enrique, Gerald, Yaswant, Fay, Simon, Angie, Marie, Laura, Colin, Steph, Vicki, Jinbo, Cath, Leyla….) por la ayuda brindada y los grandes momentos compartidos. Qué bien sentaban esas pintas en el Cuba o Ride los viernes después de una larga semana de trabajo junto a Gordon.

Al Dr. Carlos Vale y al Dr. Miguel Caetano del IPIMAR en Lisboa, por haberme introducido en la geoquímica de metales traza en el mundo del sapal. Tanto a ellos, como a toda la gente de su grupo (Joana, Rute, Patricia, João, Vasco, Marta, Pedro,…) quiero agradecer su calidad profesional y el trato personal brindado durante mi estancia en su laboratorio.

La realización de esta Tesis Doctoral ha sido posible gracias a la concesión de la beca-contrato predoctoral BFI05.52 del Programa de Formación y Perfeccionamiento de Personal Investigador del Departamento de Educación, Universidades e Investigación del Gobierno Vasco, así como a la beca de Tercer Ciclo otorgada por la Universidade de Vigo. También me gustaría agradecer al Programa Europeo Marie Curie por la concesión de una Beca para Jóvenes Investigadores en la Universidad de Liverpool. Por último agradecer a la Xunta de Galicia, en concreto a la Consellería de Innovación e Industria, por la concesión de diversas ayudas para asistencia a reuniones científicas, tanto a nivel nacional como internacional, donde he podido presentar y difundir los resultados científicos producto de mi investigación.

Esta Tesis Doctoral es una contribución al proyecto "Balance biogeoquímico y modelado 3D del transporte de metales en una ría (METRIA)", financiado por la CICYT con referencia REN2003-04106-C03, y a la Acción Complementaria para sus campañas oceanográficas, ref. CTM2004-20943-E.

i

TABLE OF CONTENTS PART I. ____________________________________________________ 1 INTRODUCTION, GENERAL METHODS AND OBJECTIVES Chapter 1. ________________________________________________________ 3 Introduction, General Methods and Objectives 1.1. Biogeochemistry of Trace Elements in Estuarine and Adjacent Waters ........... 5

1.1.1. Physical properties and gradients 1.1.1.1. Sources and mixing of dissolved salts in estuaries 1.1.1.2. Reactivity of dissolved constituents 1.1.1.3. Effect of suspended particles and chemical interactions

1.1.2. Sources and sinks of trace elements in coastal systems 1.1.3. Trace metal cycling

1.1.3.1. Sources and abundance of Trace Metals 1.1.3.2. Background on Metal Ion Chemistry 1.1.3.3. Trace Metal Cycling in the Water Column 1.1.3.4. Trace Metal Cycling and Fluxes in Sediments

1.2. Material and Methods. Trace Metal Clean Techniques and Reference Materials ........................................................................................................... 18

1.3. State of the Art. Previous Studies of Trace Elements in Galician Rias and Coastal Waters ................................................................................................. 22

1.4. Aims and Scope ............................................................................................... 23 References .............................................................................................................. 25 PART II. __________________________________________________ 35 LEVELS AND FLUXES OF TRACE METALS IN THE VIGO RIA

Chapter 2. ______________________________________________________ 37 Direct simultaneous determination of Cu, Ni and V in seawater using adsorptive cathodic stripping voltammetry with mixed ligands 2.1. Introduction ...................................................................................................... 41 2.2. Experimental .................................................................................................... 42

2.2.1. Equipment 2.2.2. Reagents 2.2.3. Analytical procedures

2.3. Results and Discussion .................................................................................... 43 2.3.1. Selection of the Optimum DMG and Cathecol Concentrations 2.3.2. Selection of the Optimum pH 2.3.3. Selection of the Optimum Adsorption Potential 2.3.4. Effect of the Adsorption Time, Working Range and Mutual Interferences 2.3.5. Accuracy, Precision and Detection Limits

ii

2.3.6. Application to Seawater Samples 2.4. Conclusions ...................................................................................................... 49 Aknowledgements References .............................................................................................................. 51 Chapter 3. ______________________________________________________ 53 Intra-annual variation and baseline concentrations of dissolved trace metals in the Vigo Ria and adjacent coastal waters (NE Atlantic Coast) 3.1. Introduction ...................................................................................................... 57 3.2. Material and Methods....................................................................................... 58 3.3. Results and Discussion .................................................................................... 59

3.3.1. Ria-Shelf spatial variation of trace metals 3.3.2. Time variation of trace metal levels along one year period

Aknowledgements References .............................................................................................................. 67 Chapter 4. ______________________________________________________ 71 Estuary-Ria exchange of trace elements in the coastal system of the Vigo Ria (NW Iberian Peninsula) 4.1. Introduction ...................................................................................................... 75

4.1.1. Study site 4.2. Material and Methods....................................................................................... 77

4.2.1. Sampling and analysis 4.2.2. Quantification of metal fluxes

4.3. Results and Discussion .................................................................................... 80 Aknowledgements References .............................................................................................................. 87

Chapter 5. ______________________________________________________ 91 Porewater Geochemistry in the Vigo Ria (NW Iberian Peninsula): Implications for Benthic Fluxes of Dissolved Trace Elements (Co, Cu, Ni, Pb, V, Zn) 5.1. Introduction ...................................................................................................... 95 5.2. Material and Methods....................................................................................... 96

5.2.1. Sample collection and pre-treatment 5.2.2. Porewater analysis 5.2.3. Quantification of sediment-water metal fluxes

5.3. Results and Discussion .................................................................................... 99 5.3.1. Geochemistry of the sediment in different areas and in different seasons 5.3.2. Metal distribution and profiles in overlying and porewaters 5.3.3. Trace metal benthic fluxes and its variation associated to the cycling of

the elements 5.4. Concluding Remarks ...................................................................................... 113 Aknowledgements References ............................................................................................................ 115

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Chapter 6. _____________________________________________________ 121 Temporal and spatial changes of total and labile metal concentration in the surface sediments of the Vigo Ria (NW Iberian Peninsula): Influence of anthropogenic sources 6.1. Introduction .................................................................................................... 125 6.2. Materials and Methods ................................................................................... 127

6.2.1. Study Area 6.2.2. Ria sampling 6.2.3. Sample pre-treatment and analysis

6.3. Results and Discussion .................................................................................. 131 6.3.1. Metal concentration and distribution in the fine fraction of Ria sediment 6.3.2. Normalization curves of background metal levels in Ria sediment 6.3.3. Spatial trends in the metal contamination of Ria sediments 6.3.4. Spatial trends in the labile metal concentrations of Ria sediments 6.3.5. Time changes in the total and labile metal of Ria sediments

6.4. Conclusion ..................................................................................................... 147 Aknowledgements References ............................................................................................................ 149 PART III. _________________________________________________ 157 DISSOLVED COPPER SPECIATION IN THE FRESHWATER DISCHARGES AND ESTUARINE MIXING OF A RIA SYSTEM

Chapter 7. ____________________________________________________ 159 Copper speciation in estuarine waters by forward and reverse titrations 7.1. Introduction .................................................................................................... 163 7.2. Material and methods..................................................................................... 164

7.2.1. Equipment 7.2.2. Reagents 7.2.3. Study Area 7.2.4. Sampling 7.2.5. Total dissolved copper determination 7.2.6. Forward titration of copper complexing ligands present in excess 7.2.7. Reverse titrations of complexing ligands already bound by copper 7.2.8. Data handling

7.3. Results and Discussion .................................................................................. 170 7.3.1. Reverse and forward titrations of copper complexing ligands

7.3.1.1. Copper 7.3.1.2. Reverse titrations of copper with SA 7.3.1.3. Forward titrations with copper

7.3.2. Comparison reverse and forward titrations 7.3.3. Ligand distribution in the Vigo Ria 7.3.4. Influence of the various ligands on copper speciation in Ria waters

Aknowledgements

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References ............................................................................................................ 177 Chapter 8. ____________________________________________________ 183 Copper speciation in continental inputs to the Vigo Ria: Sewage discharges versus river fluxes 8.1. Introduction .................................................................................................... 187

8.1.1. Study area 8.2. Sampling and Methods .................................................................................. 190

8.2.1. Sampling 8.2.1.1. Water samples

8.2.2. Copper analysis in seawater 8.2.2.1. Total dissolved copper concentrations 8.2.2.2. Copper titrations

8.2.3. Copper analysis in the SPM 8.3. Results and Discussion .................................................................................. 192

8.3.1. Master variables and SPM 8.3.2. River inputs

8.3.2.1. Levels and speciation 8.3.2.2. Fluxes: copper and ligands

8.3.3. Sewage treatment plant inputs 8.3.3.1. Levels and speciation 8.3.3.2. Sewage fluxes of copper and ligands

8.3.4. Comparison of river and sewage inputs Aknowledgements References ............................................................................................................ 205

Chapter 9. _____________________________________________________ 209 Dissolved copper speciation behaviour during estuarine mixing in the San Simon Inlet (wet season, Galicia). Influence of particulate matter 9.1. Introduction .................................................................................................... 213 9.2. Material and Methods..................................................................................... 214

9.2.1. Sampling 9.2.2. Analysis

9.3. Results ........................................................................................................... 217 9.4. Discussion ...................................................................................................... 221 Aknowledgements References ............................................................................................................ 225

PART IV. ________________________________________________ 229 IMPACT OF THE PRESTIGE OIL SPILL ON DISSOLVED TRACE METAL

LEVELS IN GALICIAN COASTAL AND OFFSHORE WATERS

Chapter 10. ____________________________________________________ 231 On the impact of the Prestige oil spill on the levels of vanadium and other trace elements inside the Vigo Ria waters (NW Iberian Peninsula)

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10.1. Introduction .................................................................................................. 235 10.2. Material and Methods................................................................................... 236 10.3. Results and Discussion ................................................................................ 237 Aknowledgements References ............................................................................................................ 241

Chapter 11. ____________________________________________________ 243 Copper, nickel, and vanadium in the Western Galician Shelf in early spring after the Prestige catastrophe: Is there seawater contamination? 11.1. Introduction .................................................................................................. 247 11.2. Material and Methods................................................................................... 248

11.2.1. Study zone 11.2.2. Sampling procedure 11.2.3. Analytical Methods

11.3. Results ......................................................................................................... 251 11.3.1. Metals in fuel 11.3.2. Total metals 11.3.3. Dissolved metals 11.3.3. Particulate metals

11.4. Discussion .................................................................................................... 254 Aknowledgements References ............................................................................................................ 257

Chapter 12. ____________________________________________________ 259 Influence of the heavy fuel spill from the Prestige tanker wreckage in the overlying seawater column levels of copper, nickel and vanadium (NE Atlantic Ocean) 12.1. Introduction .................................................................................................. 263 12.2. Material and Methods................................................................................... 265

12.2.1. Seawater samples 12.2.2. Fuel samples

12.3. Results and Discussion ................................................................................ 267 12.3.1. Changes in metal concentrations in the water column 12.3.2. Release of metals from the fuel and their dilution in seawater

Aknowledgements References ............................................................................................................ 275

SUMMARY AND GENERAL CONCLUSIONS ___________________ 279

PERSPECTIVES AND FUTURE WORK ________________________ 287 RESUMEN EN CASTELLANO _______________________________ 291

PART I

INTRODUCTION, GENERAL METHODS AND OBJECTIVES

Chapter 1

Introduction, General Methods and Objectives

1.1. Biogeochemistry of Trace Elements in Estuarine and Adjacent Waters 1.1.1. Physical properties and gradients

1.1.1.1. Sources and mixing of dissolved salts in estuaries 1.1.1.2. Reactivity of dissolved constituents

1.1.1.3. Effect of suspended particles and chemical interactions 1.1.2. Sources and sinks of trace elements in coastal systems

1.1.3. Trace metal cycling 1.1.3.1. Sources and abundance of Trace Metals

1.1.3.2. Background on Metal Ion Chemistry 1.1.3.3. Trace Metal Cycling in the Water Column

1.1.3.4. Trace Metal Cycling and Fluxes in Sediments 1.2. Material and Methods. Trace Metal Clean Techniques and Reference

Materials 1.3. State of the Art. Previous Studies of Trace Elements in Galician Rias and

Coastal Waters 1.4. Aims and Scope

References

Part I; Chapter 1

5

INTRODUCTION, GENERAL METHODS AND OBJECTIVES 1.1. Biogeochemistry of Trace Elements in Estuarine and Adjacent Waters

Estuaries are commonly described as semi-enclosed bodies of water, situated at the interface between land and ocean, where seawater is measurably diluted by the inflow of freshwater (Hobbie, 2000). Rias are estuaries which occupy former river valleys and occur on high relief coastlines (Perillo, 1995). Many of the more well studied rias are located on the Iberian Peninsula (Spain) (Castaign and Guilcher, 1995). The major difference respect to other estuaries is that in most rias the fluvial discharge is considerably weaker.

The coupling of physics and biogeochemistry occurs at many spatial scales in estuaries (Geyer et al., 2000). Estuarine circulation, river and groundwater discharge, tidal flooding, resuspension events, and exchange flow with adjacent marsh systems (Leonard and Luther, 1995) all constitute important physical variables that exert some level control on estuarine biogeochemical cycles.

Recent estimates indicate that 61% of the world population lives along the coastal margin (Alongi, 1998). These impacts of demographic changes in human populations have clearly had detrimental effects on the overall biogeochemical cycling in estuaries. Extensive growth of population and industrialization has resulted in high concentrations of inorganic contaminants (heavy metals) in estuarine sediments and waters. Fortunately, we are beginning to actually detect measurable improvements in the water and sediment quality of some estuaries (i.e. implantation of sewage treatment plants).

An understanding of the role that biogeochemical and physical processes play in regulating the chemistry and biology of estuaries is fundamental to evaluating complex management issues (Bianchi et al., 1999a; Hobbie, 2000). Biogeochemistry links processes that control the fate of sediments, nutrients, and organic matter, as well as trace metals and organic contaminants. Thus, the discipline requires an integrated perspective on estuarine dynamics associated with the input, transport, and either accumulation or export of materials that largely control primary productivity.

Introduction, General Methods and Objectives

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Biogeochemical cycles involve the interaction of biological, chemical, and geological processes that determine sources, sinks, and fluxes of elements through different reservoirs within the ecosystems.

The spatial and temporal scales of biogeochemical cycles vary considerably depending on the reservoirs considered. In the case of estuaries, most biogeochemical cycles are based on regional rather than global scales. In addition, residence time of water is different depending on the reservoir. In this way, water in the oceans has a residence time of approximately 4000 years while residence time of waters in estuaries do not exceed a month (Nace, 1971). This implies that changes and processes affecting trace elements will be observed earlier in estuaries than in the open ocean waters. 1.1.1. Physical properties and gradients

The fundamental properties of freshwater and seawater are discussed because of the importance of salinity gradients and their effects on estuarine chemistry. 1.1.1.1. Sources and mixing of dissolved salts in estuaries

Prior to discussing the factors that control concentration of dissolved components in rivers, estuaries, and the oceans, it is important to discuss the operationally defined size spectrum for different phases (dissolved, colloidal and particulate) of an element. The conventional definition for dissolved materials is the fraction of total material that passes through a membrane filter with a nominal pore size of 0.45 µm (Figure I.1.).

Estuarine environments are places where seawater is measurably diluted by freshwater inputs from the surrounding drainage basin. The mixing of river water and seawater in estuarine basins is highly variable and typically characterized by sharp concentration gradients. In simple terms, estuaries contain a broad spectrum of mixing regimes between two dominant end-members –rivers and oceans.

The sources of salts in rivers are primarily derived from the weathering of the rocks in the drainage basin of rivers and estuaries, in addition to human activities (e.g., agriculture) (Livingstone, 1963; Burton and Liss, 1976; Meybeck, 1979; Berner and Berner, 1996). Consequently, the composition of suspended materials in rivers is largely a function of the soil composition of the drainage basin. However, significant differences exist between the chemical composition of suspended materials in rivers and the parent rock material. This is due to differences in solubility of different elements in parent rock materials. For example, elements like Fe and Al are less soluble than Na and Cl, making them less abundant in the dissolved materials and more abundant in the suspended load of rivers, respectively (Berner and Berner, 1996).

Part I; Chapter 1

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Figure I.1. Conventional definition for dissolved materials shown as the fraction of total material that passes through a membrane filter with a nominal pore size of 0.45 μm. (From Wen et al., 1999)

A historical account of measurements of major dissolved components of

seawater indicate that the most abundant elements, in order of decreasing abundance are Cl-, Na+, Mg2+, SO42-, Ca2+, and K+ (Millero, 1996). In contrast to rivers, the major constituents of seawater are found in relatively constant proportions in the oceans, indicating that the residence time of these elements are long (thoushands to millions of years) highly indicative of nonreactive behaviour (Millero, 1996). In estuaries, as well as other oceanic environments (e.g., anoxic basins, hydrothermal vents, and evaporated basins), the major components of seawater can be altered quite dramatically due to numerous processes (e.g., precipitation, evaporation, freezing, dissolution, and oxidation). 1.1.1.2. Reactivity of dissolved constituents

The mixing of river water and seawater can be quite varied in different estuarine systems, resulting in a water column that can be highly or weakly stratified/mixed. These intense mixing and ionic strength gradients can significantly affect concentrations of both dissolved and particulate constituents in the water column through processes such as sorption/desorption and flocculation, as well as biological processes. Trace elements in natural waters participate in many processes which change their physico-chemical forms (speciation) or distribution in space (migration) and affect their uptake by organisms. The processes involved are oxidation/reduction, association/dissociation in solution, adsorption/desorption, precipitation/dissolution, and aggregation/disaggregation. The reactivity of a

Introduction, General Methods and Objectives

8

particular estuarine constituent has been traditionally interpreted by plotting its concentration across a conservative salinity gradient. As shown in Wen et al., (1999), the simplest distribution pattern, in a one-dimensional, two end-member, steady-state system, would be for a conservative constituent to change linearly with the salinity (Figure I.2.). For a nonconservative constituent, when there is net loss or gain in concentration across a salinity gradient, extrapolation from high salinities can yield an “effective” river concentration (C*). This “effective” concentration can be used to infer reactivity of a constituent and can be used to determine total flux of the constituent to the ocean. For example, when C*=C0, the constituent is behaving conservatively, when C*›C0, there is removal of the constituent (nonconservative behaviour) within the estuary, and when C*‹C0, the constituent is being added (nonconservative behaviour) within the estuary. River flux to the estuary and ultimately to the ocean are commonly estimated using this simplified mixing model.

Figure I.2. Illustration of the simplest distribution pattern, in a one-dimensional, two end-member, steady-state system for a conservative constituent to change linearly with salinity. (From Wen et al., 1999)

1.1.1.3. Effect of suspended particles and chemical interactions

Particulates in estuarine systems are composed by both seston (discrete biological particles) and inorganic lithogenic components. The highly dynamic character of estuarine systems (e.g., tides, wind, resuspension) can result in considerable variability in particle concentration over diurnal time intervals (Fain et

Part I; Chapter 1

9

al., 2001). Moreover, the reactivity of these particles can change over short spatial intervals due to rapid changes in salinity, pH, and redox conditions (Herman and Heipp, 1999; Turner and Millward, 2002).

Water column particulates in estuaries, primarily derived from rivers, adjacent wetland systems, and resuspension events, are important in controlling the fate and transport of trace elements in estuaries (Burton and Liss, 1976; Baskaran and Santschi., 1993; Leppard et al., 1998; Turner and Millward, 2002). A recent review by Turner and Millward (2002), with particular emphasis on metal and hydrophobic organic micropollutants (HOMs), showed that processes such as ion exchange, adsorption-desorption, absorption, and precipitation-dissolution were critical in controlling the partitioning of chemical species in estuaries (Figure I.3.). Biological processing of particulates both pelagic and benthic micro- and macroheterotrophs is also critical in estuaries.

Figure I.3. Processes critical in controlling the partitioning of chemical species in estuaries with particular emphasis on metals and hydrophobic organic micropollutants (HOMs). (Modified from Turner and Millward, 2002)

Lithogenic particles are derived from weathering of crustal materials and

mostly consist of the primary minerals quartz and feldspar, secondary silicate minerals such as clays, and hydrogenous components (Fe and Mn oxides, sulfides, and humic aggregates) formed in situ by chemical processes (Turner and Millward, 2002). Concentrations of many trace metals in estuarine waters are also influenced by sorption-desorption interactions with suspended particles (Santschi et al., 1999).

Biogenic particulates derived from fecal pellets and planktonic and terrestrial detrital materials are also important in controlling chemical interactions.

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Other suspended particulates composed of complex aggregates of biogenic and lithogenic materials have similar effects. Many of these biogenic particles are degraded and converted to dissolved organic material (DOM) which can then be sorbed to lithogenic particles, providing an organic coating. These coatings have been shown to be important in controlling the surface chemistry of particulates in aquatic environments (Loder and Liss, 1985; Wang and Lee, 1993). 1.1.2. Sources and sinks of trace elements in a coastal system

The study of the biogeochemical cycles of the elements includes the knowledge of the origin, reactivity and fate of these elements in a given environment. A conceptual representation of the biogeochemical processes in a coastal system is given in Figure I.4. In order to study the biogeochemical cycle of a given element in a coastal system it is a need to define its frontiers: the continental, atmospheric, benthic and open sea boundary. The sources (inputs) and sinks (outputs) of an element –in the dissolved and particulate phases- to a coastal system include: (a) inputs from freshwater discharges, which include rivers, streams and/or effluents (urban, industrial, domestic); (b) inputs from dry and wet atmospheric deposition; (c) outputs/inputs through porewater fluxes and particle settling/resuspension to/from the sediments and (d) exchange (inputs-outputs) with offshore waters through the open sea boundary. Within the system, the trace elements are present in different physicochemical forms (species) both in the dissolved and particulate phases, reacting with each other.

Figure I.4. Representation of the main sources and sinks of dissolved and particulate metals in a coastal system (from Cobelo-García, 2003).

WET DEPOSITION

DRY DEPOSITION

PARTICLE SETTLING AND RESUSPENSION

POREWATER

FLUXES

CONTINENTAL INPUTS

EXCHANGE WITH

OFFSHORE WATERS

ADJACENT COASTAL WATERS

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(a) Inputs from freshwater discharges. A major source of nutrients and of most trace elements to the estuaries and consequently to the ocean occurs at the land–sea interface. Rivers transport trace elements in dissolved, particulate and colloidal form. Partitioning among these phases is dependent on the properties of the element and the riverine environment. In the freshwater/seawater mixing zone, some elements are removed from solution by biological uptake and by chemical scavenging. Coagulation of colloids and small particles also contributes to trace element removal during this mixing process. Some portion of the coagulated material is buried in estuarine sediments. Desorption of particulate trace elements also takes place in this mixing zone, in part because of their displacement from particle surfaces by competition from and complexing by the major ions of seawater.

(b) Inputs from dry and wet atmospheric deposition. Atmospheric deposition is

an important, but poorly quantified, mode of transport of low-solubility trace elements from the continents to the surface waters of the estuaries and ocean. For the highly insoluble micronutrient Fe, and possibly for others such as Zn and Co, this may be the critical pathway for maintaining biologically necessary concentrations of these elements in surface waters of the open ocean. Additionally, atmospheric transport is an important vector for transferring anthropogenic materials from the continents to the coastal waters and open ocean (Duce et al., 1991).

(c) Outputs/inputs through porewater fluxes and particle settling/resuspension

to/from the sediments. Chemical fluxes between sediments and the overlying water column include net sources and sinks for dissolved trace elements in seawater as well as being a significant component of the internal cycling of trace elements in the estuaries and ocean (explained in a further part of the introduction). For some trace elements, sediments may alternately serve as a source or a sink, depending on local conditions. However, most trace elements introduced into the ocean are ultimately removed by burial in coastal and marine sediments. Diagenetic transformation of continental detritus in coastal and hemipelagic sediments may similarly release other trace elements into ocean margin waters. This is particularly true where chemically reducing conditions mobilise iron and manganese oxides formed on land, releasing oxide bound trace elements into solution (Haley and Klinkhammer, 2004). Although the release of trace elements from ocean-margin sediments has been documented for some first-row transition metals (Elrod et al., 2004; Johnson et al., 2003) the extent to which this represents a net source, by diagenetic mobilisation of continentally derived material versus the regeneration of biogenic and authigenic marine phases, remains undetermined. Several generic types of process contribute to the removal of trace elements from estuarine and seawater and their burial in marine sediments. The simplest of these is the passage of continentally derived

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particles through the water column, whether supplied by the atmosphere or by runoff, without any further solid-solution reaction. Trace elements bound within aluminosilicate minerals are included in this category. Organisms incorporate trace elements into marine particles, as do abiological solid–solution exchange reactions (e.g., adsorption, complexation). A fraction of these trace elements delivered to the seabed by sinking particles is preserved and buried in sediments. Dissolved trace elements in bottom waters may also be removed by direct sorption to surface sediments (Nozaki, 1986). Finally, precipitation from pore waters removes some dissolved trace element species that diffuse into sediments from the overlying water column. Most commonly this category of reaction is important for redox-sensitive trace elements (e.g., U, Mo, V, Re) that are soluble in the presence of oxygen but insoluble when reduced to lower oxidation states in anoxic sediments (Crusius et al.,1996). Each of the removal processes identified above is generally more active (in terms of rate per unit area) near ocean margins than in the open ocean. High biological productivity near ocean margins has an indirect effect on trace element cycles through its impact on redox conditions in underlying sediments. Low concentrations of dissolved oxygen in bottom waters coupled with high rates of respiration in surface sediments create chemically reducing conditions close to the sediment–water interface. Reduction of iron and sulphate may occur at sub-bottom depths as shallow as a few millimetres. The shallow depths of reducing conditions in sediments in contact with Oxygen Minimum Zone (OMZ) waters allows substantial fluxes across the sediment–water interface, with trace elements mobilised by reducing conditions diffusing from the sediments into the water column and species precipitated under reducing conditions moving in the opposite direction. Enhanced removal of trace elements at ocean margins, coupled with diffusive and advective exchange of water masses between shelf/slope regions and the open ocean, produces a net flux from the open ocean to ocean margins for some dissolved trace elements, a process known as boundary scavenging (Spencer et al., 1981; Bacon, 1988).

(d) Exchange (inputs-outputs) with offshore waters through the open sea

boundary. Eventually the waters reach the ocean, bearing their load of dissolved and suspended substances, and complete the hydrological cycle. The remainder material that has not been trapped in estuarine waters by being incorporated into the sediments may still be transported to the ocean, albeit in a form potentially quite different from that in which trace elements existed in freshwater. The offshore waters may also be a source of trace elements to the estuary. During upwelling events, which are typical in many costal systems around the world (i.e. Galician coastal waters), deep ocean waters reach the surface close to the continental shelf and coast contributing as an input of trace element to estuarine waters.

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1.1.3. Trace Metal Cycling

Marine biogeochemical cycles of trace elements are influenced by a complex suite of transport and transformation processes, which together are referred as ‘internal cycling’. Transformations involve trace element exchange among dissolved, colloidal and particulate forms, including the uptake of trace elements into biological material and their regeneration when this material decays. Trace elements are redistributed by coastal currents and ocean circulation, while gravitational settling of particulate material provides a unique vector transporting trace elements towards their ultimate repository in marine sediments. The importance of distinguishing between the different species lies on the fact that the different forms may assume different roles- with respect to reactivity, toxicity, transport, etc.- in the biogeochemical cycle.

1.1.3.1. Sources and abundance of Trace Metals

Like many other elements, natural background levels of trace elements exist in crustal rocks, such as shales, sandstones, and metamorphic and igneous rocks (Benjamin and Honeyman, 2000). In particular, the majority of trace metals are derived from igneous rocks in comparison with sedimentary and metamorphic rocks in the Earth´s crust. The release of trace metals from crustal sources is largely controlled by the natural forces of physical and chemical weathering of rocks, not withstanding large-scale anthropogenic disturbances such as mining, construction, and coal burning (release of fly ash). Two physical factors, the residence time and the pathways or routes along which water moves through the system, are particularly important relative to the chemical composition of natural waters (Turekian and Wedepohl, 1961).The composition of the oceans has been constant on a time scale of millions of years (Conway, 1943; Garrels and Mackenzie, 1974; Holland, 1984); however, the composition of surface waters and groundwaters continually evolves and changes on time scales of minutes to years, as these waters move along hydrologic flow paths that bring them into contact with a variety of geologic materials and biological systems (Bricker, 1987). One particularly important distinguishing feature of trace metals is their ability to bond reversibly to a broad spectrum of compounds (Benjamin and Honeyman, 2000). Thus, the major inputs of trace metals to estuaries are derived from riverine, atmospheric and anthropogenic sources.

Although trace elements typically occur at concentrations of less than 1ppb, these elements are important in estuaries because of their toxic effects, as well as their importance as micronutrients for many organisms. The fate and transport of trace elements in estuaries are controlled by a variety of factors ranging from redox conditions, ionic strength, abundance of adsorbing surfaces, and pH, just to name a few (Wen et al., 1999). The highly dynamic nature of estuarine systems, characterized by strong chemical and physical gradients, make trace metal cycling considerably more complex in estuaries compared to other aquatic systems (Morel et al., 1991; Millward and Turner, 1995). For example, the partitioning of trace metals between the dissolved and particulate fractions in estuaries can be affected by variability of in situ processes such as coagulation

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and flocculation in the estuarine turbidity maximum (ETM), resuspension events (of sediments and porewaters), and sedimentation (Figure I.5.; Santschi et al., 1997). All of these processes contribute to the complexity of trace metal speciation in estuaries (Boyle et al., 1977; Shiller and Boyle, 1987; Honeyman and Santschi, 1989; Buffle et al., 1990; Santschi et al., 1997, 1999; Wen et al., 1999). Larger scale internal and external processes such as storm events, tidal exchange, wind effects, and inputs from rivers and bordering wetland also contribute to the overall partitioning of metals in estuaries.

Figure I.5. Pathways of key processes controlling trace metal speciation in aquatic systems, as they relate to the interchange of metals between water and sediments. (Modified from Santschi et al., 1997.)

1.1.3.2. Background on Metal Ion Chemistry

While the complexation of metals with organic ligands have typically been evaluated theoretically on the basis of thermodynamic-association equilibrium models and the stability constants for the major complexes (e.g., Turner et al., 1981; Millero, 1985; Hering and Morel, 1989), this approach ignores the effects of organic ligands.

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Over the past decade, there have been numerous studies that have shown the importance of organic ligands in complexing trace metals (Sunda and Ferguson, 1983; Coale and Bruland, 1988; Santschi et al., 1997,1999), with particular emphasis on colloidal-sized particles (Benoit et al., 1994; Martin et al., 1995; Guentzel et al., 1996; Powell et al., 1996) (colloids are in the size range of 1nm to 1µm).

While dissolved and particulate inorganic compounds are clearly important in the complexation of free metals (Millward and Turner, 1995), organic complexation of metals has been shown to be a key process in estuarine waters (van den Berg, 1987; Kozelka and Bruland, 1998; Wells et al., 1998; Tang et al., 2001,2002). Overall, the distribution and speciation of trace metals in estuaries will depend on their concentrations as well as the concentrations of dissolved complexing ligands and the associated coordination sites on colloids and particulates (Kozelka and Bruland, 1998). More specifically, in the dissolved phase, the metal can occur in three different phases as: (1) free hydrated ion, (2) an inorganic complex, and (3) an organic complex.

The conditional stability constants of different functional groups in organic matter can vary significantly with different trace metals and are critical in predicting trace metal speciation.

Metal complexation with ligands is also important in controlling toxicity of metals. It has long been known that the toxicity of trace metals is more dependent upon their ionic activity than on their overall concentration (Sunda and Guillard, 1976; Anderson and Morel, 1978; Morel, 1983). As discussed earlier, factors such as pH, hardness, and DOM concentrations are key in controlling metal speciation and toxicity. Trace metals associated with colloids have been also shown to be different in their bioavailability and toxicity when compared to free aquo trace metal ions (Wright, 1977; Campbell, 1995; Doblin et al., 1999; Wang and Guo, 2000).

Interaction between particles and trace metals are also important in controlling trace metal concentrations in estuaries. For example, processes such as adsorption, desorption, flocculation, coagulation, resuspension, and bioturbation are particularly important in controlling the interaction between dissolved (free aquo) trace metals and particulates in estuaries (Santschi et al., 1997, 1999; Benjamin and Honeyman, 2000). In particular, there are important binding sites on Fe and Mn oxyhydroxides, carbonates, clays, and (particulate organic carbon) POC/ (colloidal organic carbon) COC that are essential in controlling adsorption/desorption of trace metals.

Particle-particle interactions, possibly involving metal oxides, clay minerals, and macromolecules (colloids) can have significant effects on trace metal behaviour in estuaries. One such effect, commonly referred to as the particle concentration effect (PCE) has been defined by Santschi et al., 1997 as the “physical effect which lead to decreasing overall partition coefficient with increasing particle concentration; the effect is documented for both organic and inorganic species”. The major consequences of PCE in estuaries are: (1) an increase in scavenging of trace metals at low particle concentrations, and (2) reduced desorption from particulates during resuspension events, as compared to the predicted partition coefficients of these chemical species at higher particle concentrations.

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1.1.3.3. Trace Metal Cycling in the Water Column

Trace metals that are particle reactive (e.g., Pb) or have a nutrient-like behaviour (e.g., Cd) are typically removed from surface waters via adsorption in their vertical transport through the water column. These removal processes are more likely to occur in deeper estuaries, less affected by resuspension events, where particles can become trapped at the pycnocline or redox boundary, as found in the Baltic Sea (Pohl and Hennings, 1999). It is widely accepted that hydrous oxides of Fe and Mn are important in the sorptive removal of trace metals in estuaries (Perret et al., 2000; Turner et al., 2004). The lateral and vertical distribution of these carrier-phase metals in estuaries are largely controlled by particle dynamics, as opposed to other metals (e.g., Cu, Zn and Co) which will be more affected by biotic uptake processes.

Differences in the pathways of trace metal cycling in the water column should be reflected in the overall vertical flux of particulate metals as they are transported through the water column.

Sorption-desorption from suspended particulates and sediment fluxes play a large part in controlling the nonconservative behaviour of dissolved concentrations of Fe and Mn in estuaries (Klinkhammer and Bender, 1981; Yang and Sanudo Wilhelmy, 1998).

While Fe and Mn are in many cases the ideal examples illustrating the importance of sorption/desorption processes in controlling dissolved metal concentrations, may other more bioactive metals follow similar trends, even in estuarine systems with very divergent properties (e.g., Co) (Tovar-Sanchez et al., 2004; Turner et al., 2002). While biological uptake and release of bioactive metals, such as Se, can be significant in the presence of in situ organic matter cycling (e.g., photosynthesis and respiration) (Baines et al., 2001), effects of these processes can often be masked by high loading of trace metals from anthropogenic inputs, such as occurs for Se in San Francisco Bay (Cutter and Cutter, 2004).

The distribution and speciation of trace metals across the estuarine salinity/mixing gradient has been shown to be strongly affected by the abundance of inorganic and organic material (Dai et al., 1995; Millward and Turner, 1995; Rustenbil and Wijnholds, 1996; Santschi et al., 1997). The abundance and composition of inorganic (Sholkovitz et al., 1978) and more recently organic colloids (Wells et al., 2000) have long been considered to be important in controlling trace metal behaviour.

The importance of colloidal complexation clearly varies with the trace metal in question. The destabilization of colloidal metals has been shown to be linked with the release of biopolymers from greater phytoplankton abundance in the mid-to-lower estuary, which is very different from the colloidal destabilization that occurs in the upper estuary caused by ionic changes (Wells et al., 2000). The kinetics of interactions between trace metals and different size fractions of dissolved and particulate organic and inorganic materials is essential to understand effectively the behaviour in these highly dynamic environments.

The role and number of important ligand classes that control the complexation of different trace metals is highly variable across different estuarine systems.

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To understand effectively the biogeochemistry of metal-binding ligands, it is important to be able to identify the active metal functional groups and their possible sources in estuaries. The relative importance of colloidal size in complexing trace metals may also be altered by anthropogenic changes.

As stated earlier, metal ligands may also be derived from sources other than phytoplankton, in this case ligand abundance and production may not be as tightly coupled with phytoplankton in regions receiving high riverine inputs (Shank et al., 2004). Once again, while sources of such ligands in the open ocean are likely derived directly from bacterioplankton/phytoplankton (Gonzalez-Davila et al., 1995; Moffet and Brand, 1996) or secondarily through bacterioplankton processing (Bruland et al., 1991), multiple sources of organic ligands from bacterioplankton, phytoplankton, and terrestrially derived organic matter (Bianchi et al., 2004) may be important in estuarine systems. Finally, sources of ligands from sediments may represent another important input to estuarine systems, particularly in shallow estuaries. 1.1.3.4. Trace Metal Cycling and Fluxes in Sediments

Sediments can represent sources and sinks in estuaries. Factors determining the source versus sink pathways in estuaries will largely be determined by inputs from external and in situ processing in the water column as well as postdepositional processes in sediments.

Estuarine sediments provide a long-term record of the accumulation of trace metal inputs from riverine, atmospheric, and anthropogenic sources (Kennish, 1992; Windom, 1992). In many cases, anthropogenic inputs exceed natural background levels from weathering of rock materials described earlier, because of extensive human encroachment commonly found around these areas. Thus, there needs to be a way to separate background levels from anthropogenic inputs and to account for the natural variability in composition of sediments. One method has been to normalize trace elements to a carrier phase such as Al, Fe, Li, organic carbon, or grain size (Wen et al., 1999). In the case of elemental ratios, Al has often been chosen to normalize trace element concentrations because of high natural abundance in crustal rocks and generally low concentrations in anthropogenic sources. This metal:Al ratio has effectively been used as an indicator of pollution sources in rivers and coastal systems (Windom et al., 1988; Summer et al., 1996). Down-core profiles of normalized trace metal concentrations have also been effectively used to examine historical profiles of contaminant inputs to estuaries (Alexander et al., 1993). Grain-size normalization typically involves analyzing the < 63 µm fraction since coarser grain-sized sediments (e.g., carbonates and sands) have a diluting effect on trace metal concentrations in sediments (Morse et al., 1993).

Early investigations showed trace metal concentrations in pore waters to be generally higher than in overlying bottom waters in estuarine and shallow coastal systems (Presley et al., 1967; Elderfield et al., 1981 a,b; Emerson et al., 1984). These differences result in a concentration gradient that allows trace metals in pore waters to diffuse from sediments to overlying waters (Elderfield and Hepworth, 1975). In addition to being released by diffusive mechanisms, porewater metals can become reincorporated into the sediments via adsorption, complex

Introduction, General Methods and Objectives

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formation, and precipitation (Chester, 1990, 2003). Thus the overall concentration of metals in sediments will reflect recent inputs from natural or anthropogenic sources as well as a recycling component that reflects long-term diagenetic alterations (Chester, 2003). Thus, in order to determine effectively the behaviour of metals in sediments, some assessment must be made in determining the role of sediment depositional sources and their diffusion across the sediment-water interface. In many estuarine systems, the diagenetic remobilization of metals from sediments contributes significantly to the redeposition of metals into surface sediments.

Iron and Mn cycling in estuarine sediments have been shown to be strongly linked with redox and the diagenesis of organic matter (Overnell, 2002).

While there has been considerable research on the production of metal-complexing ligands in organisms and from inputs of riverine humic substances in the water column, the potential efflux of ligands from porewaters has been largely ignored. Based on the high diffusional fluxes of DOC in some estuaries (Burdige and Homstead, 1994; Alperin et al., 1999), it is likely that ligand inputs to the bottom water column may be significant. 1.2. Material and Methods. Trace Metal Clean Techniques and Reference

Materials

Most elements are present in natural waters at concentrations below 10-5 M and are termed trace elements (Salbu and Steinnes., 1995). Because of this, special care must be taken to perform accurate and rigorous determination of trace elements since inappropriate sampling procedures, handling, storage, processing or analysis may cause sample contamination and thus overestimation of their concentrations in the environment. This is especially critical for coastal and oceanic waters as trace elements are usually in the nM-pM range (Landing et al., 1995).

Many authors have addressed the importance of using clean procedures, developing trace metal clean methodologies (Bruland et al., 1979, Boyle et al., 1981; Bewers and Windom 1982; Bruland et al., 1985, Harper, 1987) from sampling to analysis. In fact, due to ignorance on the ease of contamination during handling and / or sample analysis most of metal concentration reported before the 80´s were incorrect and significantly higher (10-1000 fold) than the values found some years later in the same areas (Kremling et al., 1983, Windom et al. 1991; Scarponi et al., 1996) using trace metal clean techniques. The start of the use of clean techniques represented a revolution regarding the analysis of trace metals in natural waters. In order to ensure the quality and accuracy of the concentrations given, a series of intercalibration programs (Geotraces, SAFe, Quasimeme) have been introduced in recent years with the aim of providing standard trace metal clean procedures for sampling, handling, storage, processing and analysis of trace elements.

The procedures used under the EPA guidelines (EPA, 1996) for sampling and analysis of trace metals in environmental samples are briefly described below and will be detailed in each chapter:

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a) Laboratory

Sample handling and analysis of trace metals in natural waters should be conducted in a clean laboratory to avoid contamination of samples from dust particles (Figure I.6.). All handling and processing of samples reported in this thesis was undertaken in a clean laboratory (Marine Biogeochemistry Group at the Institute of Marine Research, Marine Electrochemistry Group in the Department of Earth and Ocean Sciences University of Liverpool; Speciation and Environmental Analysis Group at the School of Earth, Ocean and Environmental Sciences, University of Plymouth). A clean laboratory has the peculiarity of having a positive flow of filtered air (ULPA filter of 0.2 m) through a filtration system installed in the ceiling which gives the room a Class-1000 air quality. In addition to this, handling of samples were done inside a laminar flow hood (Class-100) located in the room, reducing the risk of sample contamination.

Inside the clean room a special costume is worn, consisting of shoe-covers and a robe, both made in plastic to prevent contamination from lint of clothing and street dust accumulated in the shoes. Sample and material handling was carried out using powder-free polyethylene gloves.

Figure I.6. Clean laboratory with a filtration system on the ceiling and laminar flow hood. b) Washing and sampling procedures.

Before sampling, all material (preferably Teflon or plastic) to be used during sampling was washed to avoid contamination from walls of containers. Polyethylene (low or high density) bottles, which have been proved suitable for trace metal sampling and storage (Geotraces and EPA guidelines) were used throughout this study. The standard washing procedure was as follows: (i) first

Laminar flow hood Filtration

system

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wash in 10% HNO3 for a week, prepared from analytical grade 65% HNO3; (ii) then bottles were emptied and rinsed (x5) with Milli-Q water (Figure I.7.); (iii) bottles are then filled with pH 2 (from 65% HNO3 Merck Suprapur®) Milli-Q water; (iv) bottles are then double-bagged (zip-lock) until sampling. Other plastic material (tweezers, vials, spatulas, filter systems,...) is washed inside plastic containers in a similar way.

Figure I.7. Milli-Q water system. All water samples were collected either using 30-L Niskin bottles (X-Niskin,

General Oceanics) or with the aid of a telescopic arm for surface waters. Both methods are proposed in the protocols of clean techniques developed by the EPA. Niskin bottles were previously washed with Milli-Q water and left filled with Milli-Q at pH 2 (from HNO3 analytical grade) overnight. Before sampling collection, bottles were emptied and rinsed several times with ambient water at the sampling station before taking the final sample. A similar procedure was used when using telescopic rod. The "dirty-hands" person opened the external zip bag while the "clean-hands" wearing polyethylene gloves opened the inner zip bag to extract the polyethylene bottle (500 mL or 1 L) and re-close the bag until ready to return the bottle with the final sample. First of all, the content of the bottles (Milli-Q at pH 2 from HNO3) was emptied and bottles homogenized (x3) with the sample before collecting the final sample which was stored in a fridge (~4°C) pending filtration and analysis. In the case of sampling with telescopic rod, samples were collected facing upstream in the case of rivers and sewage treatment plants and upwind avoiding the shadow of the boat in case of surface water samples collected from the boat (EPA guidelines).

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Surface sediment samples were collected using a Van Veen grab, sub-sampling the first centimetre with a pre-washed plastic spatula. Subsequently the samples were introduced in vials previously washed in acid or zip bags and kept in refrigerator until drying and storage. Sediment cores were collected using a Rouvilloise grab with PVC cores for surface ones and a gravity corer for deeper ones. The cores were stored at 4°C until being sectioned or sliced and sub-sampled in the laboratory. c) Filtration

Filtration of samples was carried out inside a laminar flow hood in the lab clean using a vacuum pump located outside the hood to avoid contamination. Filtration systems (Nalgene) were previously acid washed (see above), whereas polycarbonate filters (Pall) were washed by immersion in 1% HCl overnight and extensively rinsed Milli-Q water before drying, weighing and storage in acid-washed petri dishes. The first 50 mL of sample were discarded after homogenization of the receiving cup; the subsequent filtrate was stored in acid pre-washed 500-1000 mL polyethylene bottles. d) Storage

Filtered samples for total dissolved metal concentrations were acidified to pH 2 (HCl Trace Select, Fluka) pending analysis, whereas those for chemical speciation were stored frozen at their ambient pH in order to avoid altering the distribution of metals among different species (Capodaglio et al., 1995).

The loaded filters were stored frozen in Petri dishes until analysis. Sediments were dried in an oven (50ºC) and sieved through 2000 and 63 µm nylon sieves. Sediments were then stored in acid-washed vials or zip-lock bags until their acid digestion prior to analysis. e) Reagents and Analysis

All reagents used for the analysis of the samples were Trace Select (Fluka) grade or Suprapur (Merck). To ensure non-contamination of samples during analysis, a series of blanks (i.e. analytical blanks) were measured. These blanks were treated in the same way as the samples (filtration, acidification, addition of reagents, etc.). Blank values were substracted to the result of each sample. Besides the analytical blanks, a series of field blanks, consisting of Milli-Q water transported to the sampling point and back to the lab with the rest of the sampling material, were also obtained. This determines the potential contamination of the sample during the sampling procedure.

Analytical methods for water, sediment and suspended particulate matter, are described in detail in each chapter. f) Certified Reference Materials

In order to check the precision and accuracy of the measurements, certified reference materials were analysed. Results appear in each chapter.

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CASS-4 (Reference Material for trace metals in coastal waters), SLRS-4 (reference material of river water for trace metals), and PACS-2 (reference material of marine sediments for trace elements) were used in this work. 1.3. State of the Art. Previous Studies on Trace Elements in Galician Rias and

Coastal Waters

As it has been reported in a recent review about the state of the knowledge of trace elements in the Galician Rias (Prego and Cobelo-García, 2003), there is still an important lack of studies on the distribution, behaviour, speciation and biogeochemical cycles of these elements in this particular environment of the coast of the Northwest Iberian Peninsula. Despite of the number of papers published in the last decade that tried to overcome this lack of information on trace metals in sediments (Cobelo-García and Prego., 2003a; Alvarez-Iglesias et al., 2003; Evans et al., 2003; Prego et al., 2006a; Marmolejo-Rodriguez et al., 2007 ), estuarine waters (Cobelo-García and Prego, 2004a; Cobelo-García et al., 2005 y Prego et al., 2006b), continental and pluvial waters (Cobelo-García and Prego, 2003b; Cobelo-García et al., 2004; Filgueiras and Prego, 2007) and organic speciation (Cobelo-García and Prego, 2004b), some aspects have not been addressed yet or suffer from scarce data.

Most part of the previous work (Prego and Cobelo-Garcia, 2003) was focused on the study of the levels of trace metal in the sediments, and although an effort has been made on the chemical speciation of metals in the different sediment fractions (Alvarez-Iglesias et al., 2003; using the sequential extraction methods: BCR or Tessier), a more complete study (wider sampling area, more metals and more fractions) is still required. In addition, there are no studies about the geochemistry of trace elements in porewaters of the sediments and benthic fluxes to the overlying waters.

Regarding trace metal studies in different estuarine or continental waters, an important advance has been made but there is still a lack of studies on trace metal distribution and baseline levels in Galician costal and open-ocean waters that will serve to evaluate the impact of any contaminant events (i.e. oil spills). Moreover, these studies were focused on the ‘classic’ elements (Cu, Pb, Zn and Cd; Cobelo-Garcia and Prego, 2004a) so an extension of the studies to a greater number of elements will highly enrich the knowledge about trace metal behaviour of trace elements in this region.

Some attempts have been made to study the chemical speciation of trace elements in Galician Rias (Cobelo-Garcia and Prego, 2004b) and a more complete work about organic speciation of trace metals in continental or estuarine waters would be beneficial.

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1.4. Aims and scope

The main objective of this Thesis is to study the main aspects about the cycle and biogeochemical processes of trace elements in the Vigo Ria and adjacent coastal waters, as well as to tackle the significance of punctual and chronic contamination. This main objective can be detailed as follows:

o Set up a fast and multielement voltammetric (Adsorptive Cathodic Stripping Voltametry; AdCSV) method valid for the analysis of trace metals in a wide type of samples (different matrices and concentrations). This method will be applied to analyze copper, nickel and vanadium in continental waters (rivers, streams and sewage treatment plants), estuarine waters, sediment porewaters, coastal and open-ocean waters.

o Establish the levels or concentrations of trace metals in the

sorroundings of the Vigo Ria, both in the dissolved and particulate fractions of the water column and in the sediment. From these data, the evolution of the contamination after the settlement of sewage treatment plants will be found out. Dissolved metals will be determined by means of the previously set up method together with the classical ASV (Anodic Stripping Voltammetry) method for the simultaneous determination of Cu, Pb, Zn y Cd in water. Regarding the surface and core sediments and the particulate matter, Atomic Absorption Spectroscopy was employed after appropriate sample acid-digestion.

o Quantify the estuarine-ria exchange of trace metals and the

importance of benthic trace metal fluxes to the Vigo Ria waters.

o Determine the organic speciation of Cu in the continental

inputs (rivers and sewage treatment plants) and its behaviour during the estuarine mixing in the Vigo Ria, paying special attention to the potential redistribution between different phases (dissolved, particulate and sediment) during mixing process.

o Evaluate the potential role of the Prestige tanker accident in

front of the Galician coasts (November 2002) on trace metal contamination in basis to the natural levels of the region.

Part I; Chapter 1

25

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